One long recognized feature of memory CD8 T cells is their continued, albeit slow, cytokine driven proliferation (so called memory turnover). Interestingly, memory CD8 T cell populations generated by experimental infection or vaccination can remain stable in numbers for long time periods in laboratory mice and recent evidence shows that memory populations are maintained for decades in humans. The stability of memory CD8 T cell populations is likely a key element of successful vaccination and much effort has been directed at understanding the cytokines (such as IL-7 and IL-15) that are essential for memory maintenance. However, these data also raise a conundrum-how are stable numbers of memory CD8 T cells maintained in the face of continued proliferation of this population? Clearly the host must have some way of counting memory cell numbers such that the proliferation of this population is balanced by equivalent death. A deeper understanding of the mechanisms controlling life and death during stable memory maintenance could not only reveal important new elements of immune system homeostasis but also provide means to enhance vaccine- induced memory or improve declining memory in the elderly. Despite the potential importance of this area, there are essentially no experimental data to address if, how and when CD8 T cells are selected for death to balance memory turnover. It seems apparent that this area of research has been stymied for lack of information identifying intermediates that are destined to die during memory turnover. Our long-term goal is to understand the molecular mediators resulting in balanced life and death during stable memory CD8 T cell maintenance. As detailed in the preliminary data we recently identified a novel CD8 T cell population representing ~20% of the memory pool. This population is characterized by the inability to produce effector cytokines such as IFN-3 in response to antigen- stimulation or to undergo substantial proliferation in response to in vivo antigen re-exposure. Importantly, we have direct evidence that this novel population can be generated from Tcm CD8 T cells during homeostatic proliferation in vivo. The immediate goal of this R21application is to test the hypothesis that this novel memory CD8 T cell population represents the T memory death intermediate (Tmdi). We will address this hypothesis through the following specific aims: Specific Aim 1. Determine if CD62LloCD27lo cytokine non-producer populations are T memory death intermediates (Tmdi). Specific Aim 2. Determine the molecular signature of putative T memory death intermediates (Tmdi). PUBLIC HEALTH RELEVANCE: Memory T cells are generated after infections or vaccination and can protect the host from reinfection with the same pathogen. Interestingly, memory T cell populations undergo continual cell division but are maintained at stable numbers for long periods of time. The goal of this proposal is to understand how memory T cell populations are counted so that this information can be used to enhance immunity after vaccination.